New Reporters of Protein Trafficking and Protein-protein Interactions in Live Cells
Author | : Marta Fernández Suárez |
Publisher | : |
Total Pages | : 297 |
Release | : 2008 |
ISBN-10 | : OCLC:260339666 |
ISBN-13 | : |
Rating | : 4/5 (66 Downloads) |
Book excerpt: (Cont.) Importantly, we showed that proximity biotinylation can detect the subcellular localization of the PPI under study. We also developed a new method for site-specific labeling of proteins in live cells. Through rational design, we re-directed E. coli lipoic acid ligase (LplA) to specifically ligate an unnatural alkyl azide substrate to an engineered 22-amino acid LplA acceptor peptide (LAP) tag. The alkyl azide can then be selectively derivatized with a cyclooctyne conjugated to any probe of interest. We first demonstrated that LplA can be used to label LAP-tagged proteins with Cy3, AlexaFluor568, and biotin at the surface of living mammalian cells, and we then applied the methodology to one- and two-color cellsurface receptor labeling. Finally, we also showed that LplA can site-specifically label intracellular proteins, although the signal/background ratio still needs to be improved.